methods. The retinas of cyclic-light–reared, pigmented arrestin knockout mice
and wild-type littermate control mice were examined histologically for
photoreceptor cell loss from 100 days to 1 year of age. In separate
experiments, to determine whether constant light would accelerate the
degeneration in arrestin knockout mice, these animals and wild-type
control mice were exposed for 1, 2, or 3 weeks to fluorescent light at
an intensity of 115 to 150 fc. The degree of photoreceptor cell loss
was quantified histologically by obtaining a mean outer nuclear layer
thickness for each animal.

results. In arrestin knockout mice maintained in cyclic light, photoreceptor
loss was evident at 100 days of age, and it became progressively more
severe, with less than 50% of photoreceptors surviving at 1 year of
age. The photoreceptor degeneration appeared to be caused by light,
because when these mice were reared in the dark, the retinal structure
was indistinguishable from normal. When exposed to constant light, the
retinas of wild-type pigmented mice showed no light-induced damage,
regardless of exposure duration. By contrast, the retinas of arrestin
knockout mice showed rapid degeneration in constant light, with a loss
of 30% of photoreceptors after 1 week of exposure and greater than
60% after 3 weeks of exposure.

conclusions. The results indicate that constitutive signal flow due to arrestin
knockout leads to photoreceptor degeneration. Excessive light
accelerates the cell death process in pigmented arrestin knockout mice.
Human patients with naturally occurring mutations that lead to
nonfunctional arrestin and rhodopsin kinase have Oguchi disease, a form
of stationary night blindness. The present findings suggest that such
patients may be at greater risk of the damaging effects of light than
those with other forms of retinal degeneration, and they provide an
impetus to restrict excessive light exposure as a protective measure in
patients with constitutive signal flow in
phototransduction.

Naturally occurring mutations that lead to a constitutive signal
flow in phototransduction have been characterized in rhodopsin,
transducin, arrestin, and rhodopsin kinase genes. Constitutive signal
flow in phototransduction is thought to underlie some forms of retinal
disorders.12345 The so-called equivalent-light hypothesis
has been proposed by Fain and Lisman24 in which
constitutive phototransduction signals are equivalent to continuous or
excessive light exposure, ultimately leading to cell death. It has been
proposed that some naturally occurring mutations leading to blindness
in humans, such as the absence of the rod photoreceptor ion
channel,4 vitamin A deficiency,2 and L296E
mutations in rhodopsin, are consistent with the
equivalent-light hypothesis because the effect of these mutations on
phototransduction simulates light exposure. However, the progression of
photoreceptor damage is difficult to track in human patients, and only
the endpoint condition is typically documented. Because of this
limitation and others (see the Discussion section), the
equivalent-light hypothesis remains to be rigorously tested under
controlled experimental conditions.

Unabated signal flow can arise from different steps in the visual
cascade. For example, certain mutations in rhodopsin can lead to
constitutive activity, especially those that affect the salt bridge
between Lys-296 and Glu-113. The interaction between Lys-296 and
Glu-113 constrains the chromophore-free opsin to an inactive
conformation.6 Disruption of this bond leads to an opsin
conformation that can support transducin activation.6 Two
known naturally occurring mutations in humans, A292E and G90D, result
in the disruption of this salt bridge by competing for the charged
residues and are thought to be responsible for causing stationary night
blindness.17 The night blindness is thought to arise from
an inability of rods, the dim-light photoreceptors, to respond to
actual light signals in the environment because of the dark–light
signals persisting from the mutant opsin.8 In transducin,
a mutation in the α-subunit in a position homologous to the oncogene
p21ras is thought to lead to prolonged
activity.5 This mutation is diagnosed in patients as the
Nougaret form of congenital stationary night blindness.5

Defects in rhodopsin shut-off can also lead to prolonged signal flow.
Rhodopsin phosphorylation by rhodopsin kinase and subsequent binding of
arrestin are necessary steps in the complete inactivation of the visual
pigment. A recessive condition called Oguchi disease is diagnosed in
patients with naturally occurring mutations that lead to nonfunctional
arrestin and rhodopsin kinase.910 Similar to the
rhodopsin A292E and G90D mutations, Oguchi disease is thought to be a
type of stationary night blindness. The implication of this clinical
diagnosis is that daytime vision remains unaffected throughout the
patient’s lifetime.

In light of recent reports that some patients with arrestin null
mutations have retinitis pigmentosa,11 it is particularly
relevant to evaluate whether constitutive signal flow due to defective
rhodopsin shut-off can cause photoreceptor cell death. We had an
opportunity to examine this issue using pigmented mice without arrestin
that we generated using homologous recombination.1213 We
have previously demonstrated that the absence of arrestin leads to
defective rhodopsin shut-off and subsequently to prolonged
photoresponse.14 Because of this defect, the rods saturate
at very low light intensities and require an excessively long time to
recover to the dark-adapted state after light exposure.14 Thus, if the defective rhodopsin shut-off and prolonged photoresponse
can lead to photoreceptor cell death, we would predict that the
arrestin knockout mice would be damaged at light levels that have no
effect on normal photoreceptors. We have now found this to be the case.
The observations have important implications for human patients with
such defects, such as those with Oguchi disease.

Materials and Methods

Mice and Lighting

The knockout allele was maintained in pigmented mice with a
mixture of 129sv and C57BL/6 genetic backgrounds, the strains used as
wild-type control mice. The wild-type and arrestin knockout
mice were born and reared in the same cyclic lighting conditions in our
laboratory (by MML), with a 12-hour light–12-hour dark cycle at an
in-cage illuminance of less than 15 fc. Some mice were reared in the
dark, and others that were cyclic-light reared to the age of postnatal
day (P) 100 were exposed to constant fluorescent light at an intensity
of 115 to 150 fc for periods of 1, 2, or 3 weeks, as described
elsewhere.15

Retinal Histology and Morphometric Analysis

The mice were killed by overdose of carbon dioxide inhalation and
immediately perfused intracardially with a mixture of mixed aldehydes
(2% paraformaldehyde and 2.5% glutaraldehyde). All procedures with
the animals adhered to the ARVO Resolution for the Use of Animals in
Ophthalmic and Vision Research and the guidelines of the University of
California San Francisco Committee on Animal Research.

Eyes were removed and embedded in epoxy resin, and histologic sections
were made along the vertical meridian.16 The tissue
sections were aligned so that the rod outer segments and Müller
cell processes crossing the inner plexiform layer were almost
continuous throughout the plane of section to ensure that the sections
were not oblique, and the thickness of the outer nuclear layer (ONL)
was measured as described elsewhere.15 Fifty-four
measurements of the ONL were made in 18 contiguous fields around the
entire retinal section (three measurements per field). These 54
measurements were either averaged to provide a single value for each
retina to allow statistical comparison of groups or plotted as a
distribution across the retina.

Results

In cyclic light, the wild-type mice showed a normal appearance
(Fig. 1a ) and no significant change in the number of photoreceptor nuclei on
the basis of ONL thickness, an index of photoreceptor
number17 at all ages up to 1 year (Fig. 2) . The arrestin knockout mice kept in cyclic light, in contrast, showed
degenerative changes as early as P100, including shorter and more
disorganized rod outer segments (Fig. 1b) than normal (Fig. 1a) . The
ONL thickness at P100 was already slightly reduced in thickness from
that in normal, wild-type mice (Figs. 1a , 1b , 2) . With increasing age,
the degeneration and loss of photoreceptors in the cyclic light-reared
arrestin knockout mice became progressively more severe (Fig. 2) . By 1
year of age, the ONL in most of the mice was reduced to less than 50%
of the normal number (Figs. 1c , 1d , 2) . In each case, from P180 to P365
the degenerative changes were more severe in the inferior than in the
superior hemisphere (Figs. 1c , 1d) .

The photoreceptor degeneration in cyclic light appeared to be caused by
light itself, because when these mice were reared in the dark, the
retinal structure was indistinguishable from that in normal wild-type
control animals (Figs. 1e , 2) . It was concluded therefore that cyclic
light causes a slow, progressive loss of photoreceptors in the arrestin
knockout mice.

To determine whether constant light would accelerate the degeneration
in arrestin knockout mice, these animals and wild-type control mice at
the age of P100 were exposed for 1, 2, or 3 weeks to fluorescent light
at an intensity of 115 to 150 fc. As expected from results in previous
studies,1213 the wild-type pigmented mice retained normal
retinal structure with no degenerative changes or loss of photoreceptor
nuclei (Figs. 1f , 23) regardless of the length of constant light exposure. The pigmented
arrestin knockout mice showed rapid photoreceptor degeneration when
exposed to constant light, with the reduction in ONL thickness of 30%
after 1 week of exposure and greater than 60% after 3 weeks of
exposure (Figs. 1g , 1h2) . The loss of photoreceptors in the arrestin
knockout mice was significantly greater in the inferior than in the
superior hemispheres of the eye (Fig. 3) .

Discussion

We have found that in pigmented arrestin knockout mice with
defective rhodopsin shut-off and prolonged
photoresponse,14 photoreceptors were progressively lost
when the animals were maintained in cyclic light. The fact that the
degeneration was prevented when the knockout mice were reared in the
dark indicates that the excessive signal flow was light mediated.

When the pigmented arrestin knockout mice were exposed to constant
light, photoreceptor degeneration was markedly accelerated. The degree
of light-induced damage in the pigmented arrestin knockout mice (Fig. 3) was almost identical with that seen in albino
mice.1518 Thus, the arrestin knockout results in a change
in susceptibility of the retina to constant light that apparently
negates the high level of protection normally afforded by eye
pigmentation.131920 The normal-pigmented control mice
were undamaged for up to 3 weeks of constant light, as expected from
results in previous studies in which similarly light-exposed pigmented
mice showed no degeneration for up to 18 weeks12 or 23
weeks.13

Another significant difference between light damage in the arrestin
knockout mice and normal albino mice is that the arrestin knockout mice
show a greater sensitivity to light in the inferior hemisphere (Fig. 3) , whereas normal albino mice are more severely damaged in the
superior hemisphere of the eye.1521

One explanation of the much greater susceptibility of the arrestin
knockout mice to excessive light and the reversal in hemispheric
sensitivity may lie in different degeneration mechanisms from those
seen in normal albino animals usually used in constant light
experiments. The main damaging agent in the nonpigmented albino eye is
thought to be reactive oxygen species.222324252627 However, it
is unlikely that significant levels of free radicals were generated
from the amount of light irradiating the retinas in the pigmented
arrestin knockout mice, given that normal pigmented mice show no damage
with up to 23 weeks of similar constant light exposure.13 Nevertheless, the amount of light entering the pigmented eye should be
sufficient to generate a signal flow that might be matched only by
bright-light exposures when normal shut-off is in place. Clearly,
direct experimental evidence is needed to ascertain the levels of
reactive oxygen species in the arrestin knockout mice, but our findings
suggest that the arrestin mouse model can allow for a functional
dissection of two molecular bases of pathogenesis: constitutive signal
flow and free radical generation.

Certain experimental results appear to be in conflict with the
equivalent-light hypothesis. For example, transgenic
mice28 and rats29 overexpressing rhodopsin
that cannot be properly turned off by phosphorylation (Ser334ter) show
photoreceptor cell loss independent of light exposure.30 Overexpression of Lys296Glu in photoreceptors of transgenic mice also
causes retinal degeneration that is apparently not related to elevated
rhodopsin activity, because it is inactivated by arrestin
binding.31 It should be pointed out that these animal
models were generated by a gene-addition technique in which the
transgene is expressed in addition to the endogenous wild-type
rhodopsin. Importantly, it has been observed that rhodopsin overdosage,
itself, can be detrimental to photoreceptors.32 The
carboxyl terminal of rhodopsin, furthermore, has been implicated in
vectorial transport of rhodopsin in photoreceptors3033 and polarized MDCK cells.34 Deletion of this domain can be
expected to disrupt rhodopsin transport and adversely affect the health
of photoreceptors through a mechanism that is unrelated to
phototransduction. These confounding variables therefore interfere with
the proper testing of the equivalent-light hypothesis. In the arrestin
knockout mice used in the present study, the only perturbation to the
system was the removal of this capping protein, leading to a defined
defect in phototransduction shut-off. Our results therefore provide
strong support to the notion that constitutive signal flow is a
stimulus for photoreceptor cell death. In other mice with a clearly
defined defect in phototransduction shut-off—that is, in rhodopsin
kinase knockout mice—constitutive signal flow appears to be a stimulus
for photoreceptor cell death.35

It has been clearly shown in the normal rat retina that the superior
hemisphere is damaged more severely by excessive light than the
inferior hemisphere, regardless of the pigmentation type or direction
of the light source.1936 Thus, some undefined intrinsic
difference exists in the two hemispheres of the rat retina in the
response to constant light, and a similar increased susceptibility of
the superior hemisphere exists in the mouse retina.1521 The significantly increased susceptibility of the inferior hemisphere
to constant light in the arrestin knockout mice also suggests that
asymmetry exists in the substrate for the degeneration. This remains to
be identified.

It is thought that cone photoreceptors are lost as a consequence of rod
cell death.3738 Because of this dependency of cones on
rod survival, both daytime vision and nighttime vision are eventually
lost, even when the primary defect lies in the rod photoreceptors. We
provide evidence that rod photoreceptors die from constitutive signal
flow that is light induced. The progression of this cell death may
eventually lead to cone loss and subsequently to total blindness, as is
evidenced in some patients with diagnosed Oguchi disease. However, we
have now found that photoreceptor cell death can be prevented by
removing the light stimulus in arrestin knockout mice. Our results
therefore provide an incentive for restricting light exposure in those
patients who have retinal disorders arising from constitutive signal
flow.

There is accumulating evidence that photoreceptors undergoing inherited
and age-related retinal degenerations may, in general, be more
susceptible to the damaging effects of excessive
light.39404142 The arrestin knockout mice, as far as we are
aware, are the most sensitive to the damaging effects of light of any
of the rodent models tested and are the first pigmented model to show
progressive retinal degeneration due simply to cyclic light exposure.
This underscores the notion that patients with mutations leading to
nonfunctional arrestin and rhodopsin kinase, such as Oguchi disease,
should avoid excessive light exposure.

Supported by National Institutes of Health Grants EY01919 and EY12155
and Core Grant EY02162 and funds from the Ruth and Milton Steinbach
Fund, Foundation Fighting Blindness, Research to Prevent Blindness, and
That Man May See. MML is a Research to Prevent Blindness Senior
Scientist Investigator.

Light micrographs of plastic-embedded wild-type and
arrestin knockout mouse retinas taken at different ages and under
different lighting conditions. All are from the posterior retina along
the vertical meridian in either the superior or inferior hemisphere.
(a) Normal retina from a wild-type mouse at P100 reared in
cyclic light with 9 to 10 rows of photoreceptor nuclei in the outer
nuclear layer (ONL), showing normal rod inner segments (RISs) and rod
outer segments (ROSs). (b) Retina from an arrestin knockout
mouse at P100 reared in cyclic light. The ONL is slightly reduced in
thickness from that in wild-type mice (a), and the ROS are
somewhat disorganized and shorter than normal (a).
(c, d) Retina from an arrestin knockout mouse
reared in cyclic light to the age of P365. The loss of photoreceptor
cells has dramatically reduced the thickness of the ONL, and the
lengths of the RISs and ROSs are significantly shorter than those in
wild-type retinas. The degeneration is more severe in the inferior
(c) than in the superior (d) hemisphere, with
fewer photoreceptor nuclei surviving in the ONL and shorter and more
disorganized RISs and ROSs. (e) The retina from an arrestin
knockout mouse appears normal after being dark-reared to the age of
P100. (f) The retina from a wild-type mouse appears normal
after it was exposed to constant light for 3 weeks. In a retina from an
arrestin knockout mouse exposed to constant light for 3 weeks, both the
inferior (g) and superior (h) hemispheres are
significantly more damaged than the wild-type retina (f).
The inferior hemisphere (g) is reduced to a single row of
nuclei and is much more severely degenerated than the superior
hemisphere (h). Toluidine blue stain. Scale bar, 25 μm.

Figure 1.

Light micrographs of plastic-embedded wild-type and
arrestin knockout mouse retinas taken at different ages and under
different lighting conditions. All are from the posterior retina along
the vertical meridian in either the superior or inferior hemisphere.
(a) Normal retina from a wild-type mouse at P100 reared in
cyclic light with 9 to 10 rows of photoreceptor nuclei in the outer
nuclear layer (ONL), showing normal rod inner segments (RISs) and rod
outer segments (ROSs). (b) Retina from an arrestin knockout
mouse at P100 reared in cyclic light. The ONL is slightly reduced in
thickness from that in wild-type mice (a), and the ROS are
somewhat disorganized and shorter than normal (a).
(c, d) Retina from an arrestin knockout mouse
reared in cyclic light to the age of P365. The loss of photoreceptor
cells has dramatically reduced the thickness of the ONL, and the
lengths of the RISs and ROSs are significantly shorter than those in
wild-type retinas. The degeneration is more severe in the inferior
(c) than in the superior (d) hemisphere, with
fewer photoreceptor nuclei surviving in the ONL and shorter and more
disorganized RISs and ROSs. (e) The retina from an arrestin
knockout mouse appears normal after being dark-reared to the age of
P100. (f) The retina from a wild-type mouse appears normal
after it was exposed to constant light for 3 weeks. In a retina from an
arrestin knockout mouse exposed to constant light for 3 weeks, both the
inferior (g) and superior (h) hemispheres are
significantly more damaged than the wild-type retina (f).
The inferior hemisphere (g) is reduced to a single row of
nuclei and is much more severely degenerated than the superior
hemisphere (h). Toluidine blue stain. Scale bar, 25 μm.

Measurements of the ONL thickness in wild-type (solid
bars) and arrestin knockout (hatched bars) mice
at different ages and under different lighting conditions. These
include mice raised in cyclic light (CyL), dark (DR), and constant
light (CL) for 1, 2, or 3 weeks. The measurements are the means ±
SD of the ONL thickness of three to eight mice of a given genotype,
age, or lighting condition. The four wild-type mice exposed to 1, 2, or
3 weeks of constant light were virtually identical (all normal; see Fig. 1f ), They were therefore pooled, and the data were plotted as the
control value for each of the CL exposure intervals. The P365 arrestin
knockout mice reared in CyL showed the largest variance, with one
having a mean ONL thickness of 30.1 μm, and the others ranging from
16.8 to 19.4 μm (one of the more degenerated retinas is shown in Figures 1gand 1h . *P < 0.05;** P < 0.001; ***P < 0.005;**** P < 0.0005 (two-tailed, unpaired t-test).

Figure 2.

Measurements of the ONL thickness in wild-type (solid
bars) and arrestin knockout (hatched bars) mice
at different ages and under different lighting conditions. These
include mice raised in cyclic light (CyL), dark (DR), and constant
light (CL) for 1, 2, or 3 weeks. The measurements are the means ±
SD of the ONL thickness of three to eight mice of a given genotype,
age, or lighting condition. The four wild-type mice exposed to 1, 2, or
3 weeks of constant light were virtually identical (all normal; see Fig. 1f ), They were therefore pooled, and the data were plotted as the
control value for each of the CL exposure intervals. The P365 arrestin
knockout mice reared in CyL showed the largest variance, with one
having a mean ONL thickness of 30.1 μm, and the others ranging from
16.8 to 19.4 μm (one of the more degenerated retinas is shown in Figures 1gand 1h . *P < 0.05;** P < 0.001; ***P < 0.005;**** P < 0.0005 (two-tailed, unpaired t-test).

Measurements of ONL thickness along the vertical meridian of the eye
from the optic nerve head (ONH) to the ora serrata (anterior margin of
the retina) in mice at P100. Mice were wild-type in cyclic light (▪),
wild-type in 1 to 3 weeks of constant light (□), or arrestin knockout
mice either in cyclic light (•) or after exposure to constant light
for 1 (⋄), 2 (▵), or 3 (○) weeks. Values are the means ± SD
of ONL thickness based on three to eight mice under each condition.

Figure 3.

Measurements of ONL thickness along the vertical meridian of the eye
from the optic nerve head (ONH) to the ora serrata (anterior margin of
the retina) in mice at P100. Mice were wild-type in cyclic light (▪),
wild-type in 1 to 3 weeks of constant light (□), or arrestin knockout
mice either in cyclic light (•) or after exposure to constant light
for 1 (⋄), 2 (▵), or 3 (○) weeks. Values are the means ± SD
of ONL thickness based on three to eight mice under each condition.

Sanyal S, Hawkins RK. Development and degeneration of retina in rds mutant mice: effects of light on the rate of degeneration in albino and pigmented homozygous and heterozygous mutant and normal mice. Vision Re. 1986;26:1177–1185.[CrossRef]

Light micrographs of plastic-embedded wild-type and
arrestin knockout mouse retinas taken at different ages and under
different lighting conditions. All are from the posterior retina along
the vertical meridian in either the superior or inferior hemisphere.
(a) Normal retina from a wild-type mouse at P100 reared in
cyclic light with 9 to 10 rows of photoreceptor nuclei in the outer
nuclear layer (ONL), showing normal rod inner segments (RISs) and rod
outer segments (ROSs). (b) Retina from an arrestin knockout
mouse at P100 reared in cyclic light. The ONL is slightly reduced in
thickness from that in wild-type mice (a), and the ROS are
somewhat disorganized and shorter than normal (a).
(c, d) Retina from an arrestin knockout mouse
reared in cyclic light to the age of P365. The loss of photoreceptor
cells has dramatically reduced the thickness of the ONL, and the
lengths of the RISs and ROSs are significantly shorter than those in
wild-type retinas. The degeneration is more severe in the inferior
(c) than in the superior (d) hemisphere, with
fewer photoreceptor nuclei surviving in the ONL and shorter and more
disorganized RISs and ROSs. (e) The retina from an arrestin
knockout mouse appears normal after being dark-reared to the age of
P100. (f) The retina from a wild-type mouse appears normal
after it was exposed to constant light for 3 weeks. In a retina from an
arrestin knockout mouse exposed to constant light for 3 weeks, both the
inferior (g) and superior (h) hemispheres are
significantly more damaged than the wild-type retina (f).
The inferior hemisphere (g) is reduced to a single row of
nuclei and is much more severely degenerated than the superior
hemisphere (h). Toluidine blue stain. Scale bar, 25 μm.

Figure 1.

Light micrographs of plastic-embedded wild-type and
arrestin knockout mouse retinas taken at different ages and under
different lighting conditions. All are from the posterior retina along
the vertical meridian in either the superior or inferior hemisphere.
(a) Normal retina from a wild-type mouse at P100 reared in
cyclic light with 9 to 10 rows of photoreceptor nuclei in the outer
nuclear layer (ONL), showing normal rod inner segments (RISs) and rod
outer segments (ROSs). (b) Retina from an arrestin knockout
mouse at P100 reared in cyclic light. The ONL is slightly reduced in
thickness from that in wild-type mice (a), and the ROS are
somewhat disorganized and shorter than normal (a).
(c, d) Retina from an arrestin knockout mouse
reared in cyclic light to the age of P365. The loss of photoreceptor
cells has dramatically reduced the thickness of the ONL, and the
lengths of the RISs and ROSs are significantly shorter than those in
wild-type retinas. The degeneration is more severe in the inferior
(c) than in the superior (d) hemisphere, with
fewer photoreceptor nuclei surviving in the ONL and shorter and more
disorganized RISs and ROSs. (e) The retina from an arrestin
knockout mouse appears normal after being dark-reared to the age of
P100. (f) The retina from a wild-type mouse appears normal
after it was exposed to constant light for 3 weeks. In a retina from an
arrestin knockout mouse exposed to constant light for 3 weeks, both the
inferior (g) and superior (h) hemispheres are
significantly more damaged than the wild-type retina (f).
The inferior hemisphere (g) is reduced to a single row of
nuclei and is much more severely degenerated than the superior
hemisphere (h). Toluidine blue stain. Scale bar, 25 μm.

Measurements of the ONL thickness in wild-type (solid
bars) and arrestin knockout (hatched bars) mice
at different ages and under different lighting conditions. These
include mice raised in cyclic light (CyL), dark (DR), and constant
light (CL) for 1, 2, or 3 weeks. The measurements are the means ±
SD of the ONL thickness of three to eight mice of a given genotype,
age, or lighting condition. The four wild-type mice exposed to 1, 2, or
3 weeks of constant light were virtually identical (all normal; see Fig. 1f ), They were therefore pooled, and the data were plotted as the
control value for each of the CL exposure intervals. The P365 arrestin
knockout mice reared in CyL showed the largest variance, with one
having a mean ONL thickness of 30.1 μm, and the others ranging from
16.8 to 19.4 μm (one of the more degenerated retinas is shown in Figures 1gand 1h . *P < 0.05;** P < 0.001; ***P < 0.005;**** P < 0.0005 (two-tailed, unpaired t-test).

Figure 2.

Measurements of the ONL thickness in wild-type (solid
bars) and arrestin knockout (hatched bars) mice
at different ages and under different lighting conditions. These
include mice raised in cyclic light (CyL), dark (DR), and constant
light (CL) for 1, 2, or 3 weeks. The measurements are the means ±
SD of the ONL thickness of three to eight mice of a given genotype,
age, or lighting condition. The four wild-type mice exposed to 1, 2, or
3 weeks of constant light were virtually identical (all normal; see Fig. 1f ), They were therefore pooled, and the data were plotted as the
control value for each of the CL exposure intervals. The P365 arrestin
knockout mice reared in CyL showed the largest variance, with one
having a mean ONL thickness of 30.1 μm, and the others ranging from
16.8 to 19.4 μm (one of the more degenerated retinas is shown in Figures 1gand 1h . *P < 0.05;** P < 0.001; ***P < 0.005;**** P < 0.0005 (two-tailed, unpaired t-test).

Measurements of ONL thickness along the vertical meridian of the eye
from the optic nerve head (ONH) to the ora serrata (anterior margin of
the retina) in mice at P100. Mice were wild-type in cyclic light (▪),
wild-type in 1 to 3 weeks of constant light (□), or arrestin knockout
mice either in cyclic light (•) or after exposure to constant light
for 1 (⋄), 2 (▵), or 3 (○) weeks. Values are the means ± SD
of ONL thickness based on three to eight mice under each condition.

Figure 3.

Measurements of ONL thickness along the vertical meridian of the eye
from the optic nerve head (ONH) to the ora serrata (anterior margin of
the retina) in mice at P100. Mice were wild-type in cyclic light (▪),
wild-type in 1 to 3 weeks of constant light (□), or arrestin knockout
mice either in cyclic light (•) or after exposure to constant light
for 1 (⋄), 2 (▵), or 3 (○) weeks. Values are the means ± SD
of ONL thickness based on three to eight mice under each condition.